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Abstract

Background

Evaluation of RNA quality is essential for gene expression analysis, as the presence
of degraded samples may influence the interpretation of expression levels. Particularly,
qRT-PCR data can be affected by RNA integrity and stability. To explore systematically
how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples
was generated by two protocols handlings of fresh tissue over a progressive time course
of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing
solution (RNAlater™) solution. Protocol B uses a dissection of placenta villosities
before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity
and stability in these two protocols according to the duration of storage.

Results

A long time tissue storage had little effect on the total RNA and mRNA integrity but
induced changes in the transcript levels of stress-responsive genes as TNF-alpha or
COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues
that underwent a dissection before RNA processing by comparison with those transferred
directly into RNA later™ solution. That loss is moderate, with average RIN (RNA Integration
Numbers) range values of 4.5–6.05, in comparison with values of 6.44–7.22 in samples
directly transferred to RNAlater™ (protocol A). Among the house keeping genes tested,
the B2M is the most stable.

Conclusion

This study shows that placental samples can be stored at + 4°C up to 48 h before RNA
extraction without altering RNA quality. Rapid tissue handling without dissection
and using RNA-stabilizing solution (RNAlater™) is a prerequisite to obtain suitable
RNA integrity and stability.

Background

Molecular tools for tissue profiling, such as real-time quantitative RT-PCR, generally
require collection of fresh frozen tissues as sources of high-quality RNA. The quality
of qRT-PCR data analysis is strongly related to the integrity and stability of the
mRNA extracted from the tissue which is in turn dependent on tissue sample processing.

The fragile nature of RNA and the question of RNases enriched tissues such as placenta
prompted us to examine the effects of storage time conditions with regard to RNA integrity
and gene expression in non fixed human term placenta.

Many parameters such as delay time, mode of tissue handling, processing protection
from RNAses degradation, tissue hypoxia, might influence the quality of extracted
RNA [1].

Time of delivery cannot be predicted with accuracy and it is quite difficult to take
in charge fresh placenta immediately after delivery. So it is difficult to standardize
the delay time from delivery to sampling. The variability of gene expression also
depends on the cellular homogeneity of the tissue. Placenta is an heterogeneous tissue
with a large pattern of different cells with foetal and maternal areas immerged in
blood. This requires a minimum of tissue washing and dissection before sampling [2].

The mode of delivery might also be important for the quality of RNA. In fact, the
duration of deliveries and the tissue hypoxia is not comparable between a placenta
excised by cesarean and a placenta that follows the vaginal tray. Is has been found
that the duration of labor might induce an hypoxia stress with a decrease of pH and
the change of the expression of a large number of genes [3].

The quality of total RNA is evaluated by the measure of its integrity and its stability.
The integrity means that the pattern of total RNA ribosomal units 28S and 18S are
abundant and that we have full length mRNA. The stability means an equal distribution
of stable housekeeping genes despite different and heterogeneous sample conditions.
It also means a stable amount of mRNA with a short half-life time.

Our purpose was to test the influence of storage of placenta at different post partum
intervals (up to 96 h every 24 h) for RNA integrity and stability, taking also in
account the mode of delivery and tissues handling before banking.

Results

Figure 1.pH and total RNA yields. pH and total placental RNA yields after storage at +4°C from 0 to 96 h (T0 to T96).
RNA samples prepared with protocol A (direct transfer to RNA later™) were compared
to RNA samples prepared with protocol B (dissection before banking). Results are expressed
as mean +/- SEM (each experiment in duplicate). (n = 14 for each protocol). p values
were determined by ANOVA. p < 0.05 was considered to be significant. pH T0 versus
T96: p = 0.002. RNA yield for protocol A versus protocol B: p = 0.01

The comparisons of pH of placenta tissue at progressive delay time showed a significant
decrease at T96 (mean pH = 6.71) compared to pH = 6.9 at T0 (Kruskall Wallis test
p = 0.002) (Fig. 1). At each delay time, pH values were found similar between vaginal and caesarean
deliveries (data not shown).

We evaluated the concentration of each total RNA extract for each delay time according
to handling protocol. Results were expressed as RNA yields (μg/mg of tissue). The
range varies from 0.14 μg/mg to 0.57 μg/mg. The overall yield was slightly but significantly
lower in protocol B (dissection of tissue before transfer) compared to protocol A
(direct transfer to RNA later™) at any time (p = 0.01) (Fig. 1). We did not notice any difference of RNA concentration between vaginal and caesarean
deliveries (data not shown).

Figure 2.Analysis of total RNA integrity according to delay time of storage and to handling
conditions. The integrity of total RNA in placental tissues stored at + 4°C for 0 to 96 H, was
determined by Agilent Bioanalyzer assay. Results (mean +/- SEM, each experiment in
duplicate) were expressed as RIN values (top of the figure), or 28S:18S ratios (bottom
of the figure). RNA was prepared from placental samples transferred directly to RNA
later™(protocol A), or dissected before banking (protocol B). p values were determined
by ANOVA. p < 0.05 was considered to be significant. RIN protocol A versus Protocol
B: p = 0.007.

Total RNA integrity was evaluated by changes of RNA Integration Numbers (RIN) and
by 28S:18S values (Fig. 2).

Mean RIN values are higher in samples extracted with protocol A (range 6.44–7.22)
compared to protocol B (range 4.50–6.05; p = 0.007). The two protocols differ from
each other with a mean delta of 1 unit of RIN. The difference was more marked at T72
and T96 (p < 0.008). No significant decrease of RIN values with delay time was observed
for the protocol A. Samples extracted with protocol B showed a significant decrease
of RIN values with delay time (p = 0.05). We did not report any difference of RIN
values between vaginal and caesarean deliveries (data not shown).

28S:18S ratios were similar in the two protocols with stable range (1.10–1.33) whatever
handling state and delay time.

Figure 3.Analysis of mRNA integrity. Comparison of FASN (a) and GAPDH (b) mRNA 5'/3' ratios in placental samples stored
at + 4°C for 0 to 96 h, as determined by qRT-PCR assays targeting sequences close
to the 5' and 3' ends of the transcripts. RNA samples prepared with protocol A were
compared to protocol B (n = 14 in each group). Results are expressed as mean +/- SEM.
Each experiment was performed in duplicate. p values were determined by ANOVA. p <
0.05 was considered to be significant. FASN 5'/3', protocol A versus protocol B (p
= 0.04), at any time of storage.

mRNA integrity was evaluated by quantification of 5' and 3' fragments of selected
large house keeping genes as fatty acid synthase (FASN) (8 kb) and glyceraldéhyde-3-phosphate
deshydrogenase (GAPDH) (3 kb). 5'/3' ratios around 1 value account for the integrity
of the transcript. A decrease of 5' fragment is predictive of mRNA degradation [4].

FASN 5'/3' ratios varied with delay time from 1.54 to 0.62 (mean +/- SEM = 1.05 +/-
0.16) in protocol A and from 0.88 to 0.31 (mean +/- SEM = 0.63 +/- 0.18) in Protocol
B (Fig. 3). The difference between the two protocols is significant (p = 0.04) assessing for
a slight degradation of 5' end of FASN gene more pronounced in protocol B. These ratios
decreased according to the delay time with a significant decrease at T72 compared
to T0 for protocol A (p < 0.05) and protocol B (p < 0.001) respectively. Ratios were
stable up to 48 h.

GAPDH 5'/3' ratios varied from 0.76 to 0.65 (mean +/- SEM 0.72 +/- 0.16) in protocol
A and from 0.91 to 0.37 (mean +/- SEM = 0.53 +/- 0.11) in protocol B (Fig. 3). The difference between the two protocols is not significant. Delay time is associated
with a significant decrease of 5'/3' ratio at T72 compared to T0 (p < 0.01) restricted
to protocol B.

Figure 4.Time course analysis of housekeeping genes. Effect of storage time and handling conditions on housekeeping gene expression.
Relative qRT-PCR amount synthetised on 1 μg of total RNA from placental tissues stored
at +4°C from 0 h to 96 h according to protocol A or protocol B (n = 14 for each group).
Reactions were normalised to contain equivalent amounts of total RNA. (a): ALAS (b):
B2M, (c): Cyclophilin. Data are plotted as mean +/- SEM. (n = 14). p values were determined
by ANOVA. p < 0.05 was considered to be significant. ALAS at T0, protocol A versus
T96, protocol B: p = 0.01. Cyclophilin, protocol A versus protocol B: p < 0.007 at
any time of storage.

Figure 5.Normalised relative abundance of TNFα and COX2 gene transcripts in placental tissues,
during post partum storage at +4°C. Geometric mean of all samples (n = 14) were normalized to the geometric mean of
B2M, ALAS, and Cyclophilin, and then relative expression was calculated using the
comparative Ct method. Results are expressed as mean +/- SEM. p values were determined
by ANOVA. p < 0.05 was considered to be significant. T96: p < 0.002.

mRNA stability was first evaluated by the comparative expression of 3 house keeping
genes: 5-aminolevulinate synthase (ALAS), β2 microglobulin (B2M), cyclophilin, according to tissue handling and delay time. These
genes are known for their stability in placenta and therefore are routinely used for
RNA normalization [5]. Fig. 4 presents the relative units in qRT-PCR obtained after correction with a calibrator.

Delay time is associated with a progressive and significant decrease of ALAS relative
values in the two protocols A and B from T0 to T96 (p = 0.007 and p = 0.03 respectively).
There was no difference according to tissue handling. Up to 72 h, (B2M relative values
were not significantly different in the two protocols with 0.62–0.81 range in protocol
A and 0.21–1.5 range in protocol B. A significant decrease of B2M expression at T96
was found for samples treated with protocol B (p < 0.01). Mean cyclophilin expression
relative values were significantly different in the two protocols with lower values
in protocol B: 0.66 +/- 0.08 (mean +/- SEM) in protocol A and 0.15 +/- 0.05 in protocol
B (p < 0.007).

The second test for RNA stability was performed only with samples extracted with protocol
A as this protocol was found to give the best stability and integrity of mRNA. Tumor
necrosis factor α (TNFα) and cyclooxygenase 2 (COX2) were chosen because of the short
life time of mRNA. Moreover, these two genes are immediate early response genes induced
by modification of tissue like hypoxia and apoptosis [6]. Our results showed a stability of TNFα and COX2 expression up to T48 followed by
a significant increase of 4 fold and 10 fold at respectively T72 and T96 (p < 0.001)
(Fig. 5).

Discussion

The limiting factor for obtaining meaningful gene expression is the quality of the
initial RNA preparation. RNA purity and integrity are of foremost importance to ensure
reliability and reproductibility of qRT-PCR [7]. Although the use of cell culture and laboratory animals allowed quick processing
of the RNA under tightly controlled protocols, this is not always the case for human
samples. It is especially true, for example, for human placenta obtained immediatly
after delivery. In the studies of placental gene expression in uncomplicated pregnancies
as well as in pregnancies complicated with diabetes, we were faced with the collection
of placentas occuring at any hour of day and night. Thereby, time course studies of
RNA expression and degradation seemed to us critically needed in order to evaluate
the biostability and quality of placental RNA species, i.e how long a placental tissue
may be stored without degradation of RNA. For some studies, the heterogeneity of the
placenta tissue requires a previous dissection of tissue to isolate the villosities
in order to study specific gene expression [8]. It was of interest to evaluate the effect on RNA yields, integrity and stability
on placenta according to handling of the tissue before banking. Therefore, we evaluated
the effects of post delivery delay time and tissue handling on RNA integrity and mRNA
expression levels.

Reliable statistical analysis of these parameters leaded us to investigate a sufficient
panel of tissues. Therefore our study was performed on 140 samples from 14 placentas,
2 protocols of preparation and 5 delay time points. Power calculations for qRT-PCR
comparisons usually indicate that a sample size of at least 50 is required to detect
difference. Hynd et al, reported that 13 cases by group yielded statistically differences
on a range of widely disparate parameters [9].

The first parameter studied was the pH of placental tissue. pH was of interest in
the light of hypoxia related to tissue injury. Hypoxia is associated with an accumulation
of lactates, a lower pH and a subsequent activation of acid lysosomial RNAses [10]. There were no consistent differences in tissue pH between placentas whatever the
mode of delivery. Tissue pH was found stable at + 4°C up to 72 h. A significant fall
of pH was found after 96 h of storage at + 4°C. This stability has been already reported
in brain tissue by others [9,11]. The overall yield of RNA was found in agreement with reports from other studies
on placenta [12]. Otherwise, the yields were lower for placentas previously dissected. This suggests
an activation of lysosomal RNAses by tissue disruption leading to a degradation of
total RNA [7-11].

The absence of significant variation according to delay time shows that degradation
of RNA seems to depend more on tissue handling than on delay time of storage. This
agrees with several reports performed on various tissues [13-17].

The assessement of total RNA integrity can be done by two main methods: the standard
28S:18S ratio and the recent RIN integration method. The standard method uses electrophoresis
of RNA and the evaluation of 28S and 18S bands and ratios. It is commonly accepted
that intact RNA has a rRNA band ratio > 1.8 [18]. We reported very stable values in all placentas samples. The more recent method
uses capillary electrophoresis and accurate integrations of peaks expressed as RIN
(RNA Integration Numbers). RIN ranges from 1 to 10 with 1 being the most degraded
profile and 10 the most intact [19]. In solid tissue, (6–8) RIN values are considered as valuable and reliable RNA [14]. Placenta samples dissected before extraction showed lower values than samples quickly
treated with RNA later™. This significant decrease of RIN values according to handling
accounts for a partial degradation of tissues by dissection. This might be explained
by the activation of intracellular RNAses during tissue disruption [20]. This agrees with corresponding RNA concentrations described in Fig. 1. Several studies reported a good correlation between RIN values and qRT-PCR relative
units [13,21]. They recommended then to consider a RIN > 6 for a suitable total RNA and RIN > 8
for a perfect RNA. Strand et al showed that RIN > 6 correlate with suitable expression
of various genes while RIN < 6 are associated with a decrease expression of these
genes [13]. Therefore, only total RNA recovered from placentas samples extracted without dissection
and stored up to 96 h in RNA-later™ may be considered as reliable for qRT-PCR.

Our observations suggest that despite the 28S:18S ratio is considered as the gold
standard for the measure of integrity, it lacks precision and discrimination between
preserve and partially degraded RNA.

Following total RNA integrity, determination of mRNA integrity is important to assess.
We analysed it by the quantification of 5' and 3' ends fragments of some gene transcripts.
The fragments located towards the 5'end of the mRNA of housekeeping genes are used
as indicator sequences for the degree of degradation [17,22].

FASN 5'/3' observed ratios are higher than those reported by Bauer in blood samples
[22]. This might be explained by a high stability of 5' ends of genes expressed in placenta
tissues [23]. FASN and GAPDH 5'/3' ratios are higher in protocol A compared to protocol B and
are probably related to a partial degradation of mRNA after dissection. Expression
levels of FASN and GAPDH fragments were stable up to 48 h when samples were kept at
4°C. This shows that the delay of storage has an effect on the integrity of mRNA after
48 h. This effect is higher in protocol B and fits with the decrease of total RNA
integrity measured by RIN. Previous studies have observed intact total RNA in various
tissues stored at + 4°C post mortem, such as human brain (up to 36 h), human bone.(up
to 48 h) liver of rabbit (up to 96 h) and bovine muscle (up to 8 days) [15,17,24]. This confirms the high stability of RNA in most of tissues when stored at low temperature.
The delay time has a less effect on GAPDH expression in samples treated with protocol
A. In fact, we obtain very stable ratios compared to those observed with FASN. The
difference of lengths of FASN and GAPDH transcripts respectively 8 kb and 3 kb might
explain this difference. A very long transcript is more sensitive to partial degradation
than a smaller one.

Endogenous controls, usually housekeeping genes, are measured to better normalize
between tissue samples [25-28]. The choice of good controls is tissue dependant, and the same housekeeping genes
suitable for a tissue are not for another [27-29]. Previous studies have compared a set of housekeeping genes in placenta by qRT-PCR
[5]. B2M, ALAS and cyclophilin have been reported as stable genes in placenta and so
far used in this study [5]. Our observations highlight variability of expression profiles for these 3 genes
according to handling and/or delay time. B2M appears as the most stable gene no sensitive
to conditions of storage or tissue handling. This agrees with a previous study showing
that B2M is one of the most stable housekeeping gene in placenta [5]. ALAS mRNA expression is sensitive to storage and cyclophilin to tissue handling.
This seems not to depend on the length of transcripts that are quite similar for these
3 genes. It is important to note that the length of the amplicon is over 200 bp for
cyclophilin and about 100 bp for B2M and ALAS. Others reported a correlation between
total RNA integrity measured by RIN and the efficiency of qRT-PCR according to the
length of the PCR product [21,30]. Taken together, these results highlight that storage and handling influence the
expression of standard housekeeping genes in placentas. B2M was found the most stable
gene in placentas stored up to 48 h whatever the mode of preparation.

The analysis of TNFα and COX2 mRNA PCR products show a stability of expression up
to 48 h and thereafter an important up regulation (4 fold and 9 fold at interval 72
h and 96 h respectively). TNFα and COX2 are involved in cellular defense and stress
response. Overexpression of these genes induced by storage of rat liver at 37°C has
been already reported [24]. The mechanism may be a stabilization of labile mRNA through, for example the activation
of MAPK or other signaling pathways [31]. This activation of signaling pathways might be enhanced by ischemia and apoptosis
of tissues during a long period of storage. Despite the little effect of delay time
of storage on RNA integrity, our observation shows that it is important to take into
account these variations of expression of inducible genes.

Conclusion

This study, from the criteria of RNA yields, global RNA integrity, and RNA expression
of some stable and some unstable mRNA, shows, for the first time, that it is routinely
possible to obtain RNA of good quality from placentas stored intact at +4°C up to
48 h and transferred into a RNA stabilizing solution. However, one must be cautious
for an extrapolation to placental expression of all the genes. Nevertheless, the delay
time will be helpful for RNA preparation from this tissue, as an immediate processing
is not always easy to plan. Dissection of placentas in order to obtain a tissue free
of vessels and foetal membranes must be avoided or set up using a RNA-stabilizing
solution.

Our results are in agreement with those from other previous studies on various human
and animal tissues showing that RNA degradation is a minor problem when intact tissues
are stored either at +4°C or even at room temperature before biobanking.

Methods

Sampling, tissue Handling

Placentas at full term (38–41 weeks of gestation), were obtained after caesarean sections
or normal vaginal deliveries from 14 women with healthy babies and no complications
of pregnancy. All the placentas from the patients were collected from one maternity
hospital (CHRU Lille, Hôpital Jeanne de Flandre). This study is a preparative part
of a PHRC approved by the Ethics Committee of LILLE (France), CPP (Comité Consultatif
de Protection des Personnes dans la Recherche Biomédicale). Data were recorded anonymously.
Placentas were stored at +4°C until analysis.

Biopsy placental villi (about 1 cm3) were removed and processed after storage of placenta at various delay times (0 h,
24 h, 48 h, 72 h, 96 h). For each placenta, tissue fragments were obtained from 4
various locations between the decidual and chorionic plates in order to limit the
tissue heterogeneity [8], then submitted to 3 different treatments: (1) 150 mg of tissues were transferred
immediately in a tube containing 2 ml of a commercial RNA-stabilizing solution (RNAlater™,
Qiagen, SA), stored at +4°C overnight submitted to 3 different treatments: (1) 150
mg of tissues were transferred immediately in a tube containing 2 ml of a commercial
RNA-stabilizing solution (RNAlater™, Qiagen, SA), stored at +4°C overnight and then
moved to -80°C for long time storage (protocol A), (2) tissues were briefly washed
in sterile 100 mM CaCl2 and PBS in order to remove blood, dissected in Petri dishes, made free of foetal membranes
vessels and tissue from maternal origin, rinsed in sterile PBS solution, then adjusted
to 150 mg, transferred to RNA later™ and stored at -80°C until processed (protocol
B). (3) tissues were transferred to a tube containing 5 volumes of water, disrupted
with a Qiagen tisssue disrupter, and the supernatant transferred to a new tube to
measure the pH. All the experiments were performed on ice excepted tissue preparation
for pH determination.

RNA extraction

Each frozen sample (stored at -80°C in RNAlater™) was placed in a 13 ml tube containing
4 ml of lysing buffer solution (Qiagen, RNA easy midi Kit ™). Samples were homogenized
with a Qiagen tissue disrupter using two 20–30 sec pulses, and processed for RNA isolation
using the RNAeasy midi kit™ followed by an additional treatment with DNAse according
to manufacturer's procedure (Qiagen). RNA concentration was evaluated by measuring
the absorbance at 260 nm using the spectrophotometer Nanodrop. RNA samples extracted
from placental tissues obtained from 4 various locations (see above) were pooled.
Aliquots of 1 μg of pooled RNA samples were precipitated in ethanol for long storage.
All tubes were RNAse free.

RNA microchip electrophoresis

Structural RNA integrity was evaluated using a microchip electrophoresis on an AGILENT
2100 Bioanalyser (Agilent technologies) [32]. All chips (RNA6000 labchips kit) were prepared and loaded according to the manufacturer's
instructions. The results were displayed as gel-like images and electrophoregrams.
Total RNA degradation was evaluated using 2 criteria (1) the RNA integrity number
(RIN) (2) the decrease in 28S and 18S peak areas [18] (see Additional file 1).

Additional file 1.Electrophoretic tracings of RNA according to different handling methods. the data provived presented AGILENT graphs of two RNA samples extracted from the
same tissue and submitted to Protocol A: RIN 7.2 value (a) and protocol B: RIN 4.5
value (b).

cDNA synthesis

Reverse transcription was performed using the instructions for the cDNA kit (Invitrogen,
ThermoScript RT-PCR system). A 20 μl reaction without reverse transcriptase was performed.
A negative control without RNA was included in the reverse transcription reaction.
1 μg of total RNA was reverse transcribed at 50°C for 45 min with a mixture containing
4 μl 5 × cDNA RT buffer, 15 U Thermoscript reverse transcriptase, 1 mM dNTPs, 2.5
μM oligo-dT20 anchored primer, 40 U Rnase out and 5 mM DTT in a final volume of 20 μl.

qRT-PCR analysis

Relative expression levels of RNA per sample were quantified by SYBR Green I assay
on Roche Light Cycler 2700 sequence detection assay (Meylan, France). For each transcript,
PCR was performed in duplicates with 10 μl reaction volumes of 1 μl of cDNA, 8 μl
of mix, and 1 μl of each primer set. PCR was conducted using the following cycle parameters:
2 min at 50°C, 10 min at 95°C and 40 three steps cycles of 15 sec at 95°C, 20 sec
at 50°C and 20 sec at 72°C. The assay was performed following the manufacturer's recommendations
except that the reaction volume was reduced to 10 μl. A pool of cDNA from control
placenta tissues prepared immediately after partum was used as a standard (in threefold
serial dilutions) for quantitative correction. All cDNA samples were applied in dilution
of 1:5 to obtain results within the range of the standard. Each sample was evaluated
in duplicate. Analysis of transcript level was carried out using first the determination
of the threshold cycle Ct for each reaction corrected by the efficiency. Then the
delta Ct was calculated by subtracting the mean Ct of the calibrator from each value
of Ct for each gene. The amount of target relative to a calibrator was computed by
2 -delta Ct

cDNA evaluation of integrity

cDNA integrity was investigated by comparing the qRT-PCR 5'/3' ratio for 2 selected
genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthase (FASN).
These two genes were chosen because of their ubiquitous expression as a so-called
house keeping (HK) genes and for their various sizes, short for GAPDH (1 kb) and large
for FASN (>8 kb).

Two primer pairs generating amplification products of different sizes were spaced
at 5' and 3' ends along the FASN cDNA generating fragments of 197 and 281 bp [22]. Two primers pairs for GAPDH cDNA were designed using primer3 software http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgiwebcite that generates fragments located at 3' and 5' ends of cDNA (Table 1). This method takes advantage of the fact that the oligo-dT primed cDNA population
contains complementary DNA that extends from the 3'-end to the 5'-mRNA cap structure
in intact mRNA. The 5'end of the mRNA will be underrepresented in the cDNA population
to a degree corresponding to the extent of degradation in the RNA preparation. The
relative yields of the amplification products from 5' end to 3' end of the mRNA may
be used as a relative measure of the fraction of fragmented versus intact FASN and
GAPDH mRNA in the sample. All FASN and GAPDH primer binding sites were located on
different exons so that avoiding contaminating genomic DNA. Results were expressed
as 5'/3' ratio of relative values obtained for each gene fragment.

mRNA evaluation of stability

Stability of mRNA was evaluated by quantification of a first set of selected HK genes
almost stable: 5-aminolevulinate synthase (ALAS), β2 microglobulin (B2M), and cyclophilin, and a second set of 2 genes with a short half
life: TNFα (37 min) and COX2 (3 h). Primers were designed by Qiagen (GENGLOBE) for
ALAS and B2M or by primer3 software for cyclophilin (Table 1). TNF and COX2 relative expression were normalised by subtracting the mean geometric
Ct of the 3 HK genes to the each Ct using geNorm software. Results were expressed
as 2 -deltadeltaCt [33].

Statistical analysis

To examine whether the variable qRT-PCR amounts, RNA concentrations, RIN, pH values
28S:18S ratios, were different between the groups defined by pre analytical conditions
and the delay time of conservation, we used either the Kruskal-Wallis test (3 groups
minimum) or the Mann-Whitney test (2 groups) which are non parametric alternative
to one-way Anova.

All statistical analyses were performed using ABI PRISM software. p values less than
0.05 were considered statistically significant.

Authors' contributions

IF designed the study, carried out the analysis and interpretation of the results
and drafts the manuscript, EM contributed to the development of methodology and performed
the experimental procedure, AV and PD provide placenta tissues and are physicians
involved in a common clinical research project on foetal growth and gene expression
in placenta, MVM assisted in the preparation of RNA. JR participated in the conception
and design of the study and helped to draft the manuscript. All authors read and approved
the final manuscript.

Acknowledgements

The authors would like to thank Céline Villenet and Jean Pierre Kerckaert (IFR INSERM114-IMPRT)
for AGILENT technology assistance, Pia-Manuela Voicu for English language corrections,
nurses and obstetrician team of Hôpital Jeanne de Flandre for placenta recruitment.